Stable non-aqueous single phase viscous vehicles and formulations utilizing such vehicle

ABSTRACT

This invention relates to stable non-aqueous single phase viscous vehicles and to formulations utilizing such vehicles. The formulations comprise at least one beneficial agent uniformly suspended in the vehicle. The formulation is capable of being stored at temperatures ranging from cold to body temperature for long periods of time. The formulations are capable of being uniformly delivered from drug delivery systems at an exit shear rate of between about 1 to 1×10 −7  reciprocal second.

This Application claims the benefit of 60/119,170, filed Feb. 8, 1999.

FIELD OF THE INVENTION

This invention relates to stable non-aqueous single phase biocompatibleviscous vehicles capable of suspending beneficial agents and uniformlydispensing said agents at low flow rates and more particularly to stableuniformly mixed formulations of beneficial agents in stable non-aqueoussingle phase biocompatible viscous vehicles.

REFERENCES

The following references are referred to by numbers in brackets ([])atthe relevant portion of the specification.

-   -   1. Wang, et al., J. Parenteral Sci. Tech, 42: S4-S26 (1988).    -   2. Desai, et al., J. Am. Chem. Soc., 116: 9420-9422 (1994).    -   3. Chang, et al., Pharm. Tech., 80-84 (Jan. 1996).    -   4. Manning, et al., Pharm. Res., 6: 903-918 (1989).    -   5. Hageman, Drug Dev. Ind. Pharm, 14: 2047-2070 (1988).    -   6. Bell, et al., Biopolymers, 35: 201-209 (1995).    -   7. Zhang, et al., Pharm. Res. 12: 1447-1452 (1995).    -   8. PCT published application 98/00158    -   9. PCT published application 98/16250    -   10. Knepp, et al., Pharm. Res. 15 (7) 1090-1095 (1998).    -   11. PCT published application 98/00157    -   12. PCT published application 98/00152    -   13. U.S. Pat. No. 5,540,912    -   14. U.S. Pat. No. 5,571,525    -   15. U.S. Pat. No. 5,512,293    -   16. PCT published application 96/40049    -   17. Yu, et al., J. Pharm. Sci., 85: 396-401 (1996).    -   18. Mitchell, U.S. Pat. No. 5,411,951 (1995).    -   19. Brooks, et al., U.S. Pat. No. 5,352,662 (1994)    -   20. Geller, L., U.S. Pat. No. 3,869,549 (1975).    -   21. Larsen, et al., PCT Publication No. W095/34285 (1995).    -   22. Knepp, et al., J. Pharm. Sci. Tech, 50: 163-171 (1996).    -   23. U.S. Pat. No. 5,614,221    -   24. U.S. Pat. No. 4,594,108    -   25. U.S. Pat. No. 5,300,302    -   26. U.S. Pat. No. 4,588,614    -   27. U.S. Pat. No. 4,310,516    -   28. U.S. Pat. No. 5,635,213    -   29. EP 379,147

BACKGROUND OF THE INVENTION

Peptides, polypeptides, proteins and other proteinaceous substances(e.g., viruses, antibodies) collectively referred to herein as proteins,have great utility as pharmaceuticals in the prevention, treatment anddiagnosis of disease. Proteins are naturally active in aqueousenvironments, thus the preferred formulations of proteins have been inaqueous solutions. However, proteins are only marginally stable inaqueous solutions. Thus, protein pharmaceuticals often have shortshelf-lives under ambient conditions or require refrigeration. Further,many proteins have only limited solubility in aqueous solutions. Evenwhen they are soluble at high concentrations, they are prone toaggregation and precipitation.

Because proteins can easily degrade, the standard method for deliveringsuch compounds has been daily injections. Proteins can degrade via anumber of mechanisms, including deamidations of asparagine andglutamine; oxidation of methionine and, to a lesser degree, tryptophan,tyrosine and histidine; hydrolysis of peptide bonds; disulfideinterchange; and racemization of chiral amino acid residues [1-7]. Wateris a reactant in nearly all of these degradation pathways. Further,water acts as a plasticizer, which facilitates unfolding andirreversible aggregation of proteins. Since water is a participant inalmost all protein degradation pathways, reduction of aqueous proteinsolution to a dry powder provides an alternative formulation methodologyto enhance the stability of protein pharmaceuticals.

One approach to stabilizing proteins is to dry them using varioustechniques, including freeze-drying, spray-drying, lyophilization, anddesiccation. Dried proteins are stored as dry powders until their use isrequired.

A serious drawback to drying of proteins is that often one would like touse proteins in some sort of flowable form. Parenteral injection and theuse of drug delivery devices for sustained delivery of drug are twoexamples of the applications where one would like to use proteins in aflowable form. For injection, dried proteins must be reconstituted,adding additional steps which are time-consuming and where contaminationmay occur, and exposing the protein to potentially destabilizingconditions [7]. For drug delivery devices the protein formulations mustbe stable for extended periods of time at body temperature and maintaintheir flowability for the expected life of the device.

Solution formulations of proteins/peptides in non-aqueous polar aproticsolvents such as DMSO and DMF have been shown to be stable at elevatedtemperatures for long periods of time [8]. However, such solvent basedformulations will not be useable for all proteins since many proteinshave low solubility in these solvents. The lower the solubility of theprotein in the formulation, the more solvent would have to be used fordelivery of a specific amount of protein. Low concentration solutionsmay be useful for injections, but may not be useful for long termdelivery at low flow rates.

Proteins have been formulated for delivery using perfluorodecalin [9,10], methoxyflurane [9], high concentrations in water [11], polyethyleneglycol [12], PLGA [13, 14], ethylenevinylacetate/polyvinylpyrridonemixtures [15], PEG400/povidone [16]. However, these formulations werenot shown to retain a uniform suspension of protein in viscous vehicleover long periods of time.

Many biologically active compounds degrade over time in aqueoussolution. Carriers in which proteins do not dissolve but rather aresuspended, can often offer improved chemical stability. Furthermore, itcan be beneficial to suspend the beneficial agent in a carrier when theagent exhibits low solubility in the desired vehicle. However,suspensions can have poor physical stability due to settling andagglomeration of the suspended beneficial agent. The problems withnon-aqueous carriers tend to be exacerbated as the concentration of theactive compound is increased.

Dispersing powdered proteins or peptides in lipid vehicles to yieldparenteral sustained release formulations has been investigated [17-21].The vehicles used were either various vegetable (sesame, soy, peanut,etc. ) or synthetic oils (e.g., Miglyol) gelled with aluminum fatty acidesters such as aluminum stearates (mono-, di- or tri-), or with apolyglycerol ester. Although theoretically these vehicles might precludesolution denaturation and protect the drug from aqueous chemicaldegradation, the vehicles themselves are unstable at highertemperatures. The storage of liquid vegetable oils at body temperaturesresults in the formation of reactive species such as free fatty acidsand peroxides (a process which is accelerated by the presence of tracesof various metal ions such as copper or iron which can leach from someimplantable devices). These peroxides not only adversely affect proteinstability [22] but would be toxic when delivered directly to, forexample, the central nervous system of a human or animal.

The sustained delivery of drugs has many advantages. Use of implantabledevices assures patient compliance, since the delivery device istamperproof. With one insertion of a device, rather than dailyinjections, there is reduced site irritation, fewer occupational hazardsfor practitioners improved cost effectiveness through decreased costs ofequipment for repeated injections, reduced hazards of waste disposal,and enhanced efficacy through controlled release as compared with depotinjection. The use of implantable devices for sustained delivery of awide variety of drugs or other beneficial agents is well known in theart. Typical devices are described, for example, in U.S. Pat. Nos.5,034,229; 5,057,318; 5,110,596; and 5,782,396. The disclosure of eachof these patents is incorporated herein by reference.

For drug delivering implants, dosing durations of up to one year are notunusual. Beneficial agents which have low therapeutic delivery rates areprime candidates for use in implants. When the device is implanted orstored, settling of the beneficial agent in a liquid formulation canoccur. This heterogeneity can adversely affect the concentration of thebeneficial agent dispensed. Compounding this problem is the size of theimplanted beneficial agent reservoir. Implant reservoirs are generallyon the order of 25-250 μl, but can be up to 25 ml.

Viscous formulations have been prepared using two separate components tobe mixed with drug at use [23], thickening agents added to aqueouscompositions [24], gelling agents added to aqueous drug solutions [25],porous textile sheet material [26], thickening agents with oleaginousmaterial [27], viscous aqueous carrier for limited solubility drug [28],and extrudable elastic gels [29]. However, these formulations are mixedat use, contain aqueous components, use sheet matrices, or are deliveredtopically, orally, or intraduodenally.

Stability of formulations can be enhanced by freeze-drying, lyophilizingor spray-drying the active ingredient. The process of drying the activeingredient includes further advantages such as compounds which arerelatively unstable in aqueous solution can be processed and filled intodosage containers, dried without elevated temperatures, and then storedin the dry state in which there are relatively few stability problems.

Pharmaceutical formulations, particularly parenteral products, should besterilized after being sealed in the final container and within as shorta time as possible after the filling and sealing have been completed.(See, for example Remington, Pharmaceutical Sciences, 15^(th) ed.(1975)). Examples of sterilization techniques include thermal ordry-heat, aseptic, and ionized radiation. Combinations of thesesterilization procedures may also be used to produce a sterile product.

There is a need to be able to deliver protein compositions to the bodywhich are stable at body temperatures over extended periods of time toenable long term delivery of the protein. There is a need to be able todeliver concentrations of proteins that are efficacious. There is a needfor a novel non-aqueous formulation capable of homogeneously suspendingproteins and dispensing such agents at body temperatures and low flowrates over extended periods of time.

SUMMARY OF THE INVENTION

The present invention provides stable single phase non-aqueousbiocompatible viscous vehicles capable of forming uniform suspensionswith proteins. The components of the viscous vehicle comprise at leasttwo of polymer, surfactant, and solvent. The ratios of the componentswill vary depending on the molecular weight of the components and thedesired viscosity of the final vehicle. Presently preferred componentratios are: polymer, about 5% to about 60%; solvent, about 30% to about50%; and surfactant, about 5% to about 20%.

The present invention also provides stable formulations in whichbeneficial agents are uniformly suspended in stable single phasenon-aqueous biocompatible viscous vehicles. In particular, thebeneficial agents are formulated in the viscous vehicles atconcentrations of at least about 0.1 %, depending upon the potency ofthe beneficial agent. These stable formulations may be stored at thetemperature appropriate for the beneficial agent, ranging from cold, tobody temperature (about 37° C.) for long periods of time (1 month to 1year or more). In a preferred embodiment the formulation comprises about0.1 to 50% (w/w) of beneficial agent, depending on the potency of thebeneficial agent, the duration of treatment, and the rate of release forthe drug delivery system.

These formulations are especially useful in implantable delivery devicesfor long term delivery (e.g., 1 to 12 months or longer) of beneficialagent at body temperature, preferably about 37° C. Thus, the presentinvention also provides for the delivery of said proteins to the bodyover extended period of time to enable long term delivery of the proteinat low flow rates of about 0.3 to 100 μl/day, preferably about 0.3 to 4μl/day for about a 6 month delivery period and preferably 5 to 8 μl/dayfor about a 3 month delivery period.

In another aspect, the invention provides methods for preparing stablenon-aqueous biocompatible formulations of a beneficial agent in a singlephase viscous vehicle. Preferred formulations comprise about 0.1 to 50%(w/w) beneficial agent depending on the potency of the beneficial agent,the duration of treatment, and the rate of release from the deliverysystem.

In yet a further aspect, the invention provides methods for treating asubject suffering from a condition which may be alleviated byadministration of a beneficial agent, said methods comprisingadministering to said subject an effective amount of a stablenon-aqueous formulation comprising at least one beneficial agentuniformly suspended in a single phase viscous vehicle.

A further aspect of the invention is that non-aqueous single phaseviscous vehicles containing beneficial agents are chemically andphysically stable over a broad temperature range for long periods oftime. The beneficial agents in the viscous vehicles are also chemicallyand physically stable over a broad temperature range for long periods oftime. Thus, these formulations are advantageous in that they may beshipped and stored at temperatures below, at, or above room temperaturefor long period of time. They are also suitable for use in implantabledelivery devices in which the formulation must be stable at bodytemperature for extended periods of time.

The formulations of the present invention also remain stable whendelivered from implantable drug delivery systems. The beneficial agentshave been shown to exhibit zero order release rates when delivered fromimplantable drug delivery systems at very low flow rates over extendedperiods of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of human growth hormone (hGH) formulations ofthe present invention as determined at 37° C. by reverse phase HPLC.

FIG. 2 shows the stability of hGH formulations of the present inventionas determined at 37° C. by size exclusion chromatography.

FIG. 3 shows the average release rate (μg/day) of 10% (w/w) spray-driedlysozyme in formulations of the present invention.

FIG. 4 shows the average release rate (μg/day) of 10% (w/w) spray-driedhGH in a glycerol monolaurate/lauryl lactate/polyvinylpyrrolidonevehicle.

FIG. 5 shows the average release rate (μg/day) of 10% lysozyme in alauryl alcohol/polyvinylpyrrolidone vehicle.

FIG. 6 shows the average release rate ((μg/day) of 25% lysozyme in aglycerol monolaurate/lauryl lactate/polyvinylpyrrolidone vehicle.

FIG. 7 shows the average release rate ((μg/day) of 33% lysozyme in aglycerol monolaurate/lauryl lactate/polyvinylpyrrolidone vehicle.

FIG. 8 shows the average release rate ((μg/day) of 45% lysozyme in aglycerol monolaurate/lauryl lactate/polyvinylpyrrolidone vehicle.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to the unexpected discovery thatuniformly suspending beneficial agents in non-aqueous single phasebiocompatible viscous vehicles results in stable formulations which canbe delivered at body temperature over an extended period of time at lowflow rates. Previously known formulations of beneficial agents which arebuffered aqueous or non-aqueous solutions which may or may not containexcipients do not provide formulations which can be uniformly dispensedat body temperatures at low flow rates over an extended period of timewithout exhibiting unacceptable amounts of aggregation or degradation ofthe formulation. The presently claimed formulations stabilize beneficialagents and can be stored at the temperature appropriate for thebeneficial agent. The temperatures can range from cold (not exceeding 8°C.) to body temperature (about 37° C.) for long periods of time. Theseformulations are especially useful in implantable delivery devices forlong term delivery (e.g., 1 to12 months or longer) of drug at low flowrates and at body temperature, preferably about 37° C.

Standard beneficial agent formulations consist of dilute aqueous ornon-aqueous solutions or suspensions. Drug stability is usually achievedby varying one or more of the following: pH, buffer type, ionicstrength, excipients (EDTA, ascorbic acid, etc.) For these formulations,degradation pathways requiring water (hydrolysis, deamidation,racemization) cannot be fully stabilized. In the present invention,beneficial agents formulated in non-aqueous biocompatible single phaseviscous vehicles containing for example, polyvinylpyrrolidone, vinylacetate, and/or polyoxyethylenepolyoxypropylene block copolymers wereshown to be chemically and physically stable. The viscosity of theformulation will depend upon a number of criteria, including thebeneficial agent potency and concentration, and the process by which theformulation is prepared. The viscosity of the formulation can be chosenso that the desired amount of beneficial agent is delivered over thedesired period of time.

The invention also consists of non-aqueous single phase biocompatibleviscous vehicles capable of uniformly suspending beneficial agents andformulations containing at least one beneficial agent uniformlysuspended in said viscous vehicle. The invention also consists offormulations containing at least one beneficial agent uniformlysuspended in a non-aqueous single phase biocompatible viscous vehicle,which formulations are stable for an extended period of time at bodytemperatures, and capable of delivering said beneficial agents uniformlyat low flow rates. The discovery consists of the realization that stablenon-aqueous viscous vehicles improve the stability of beneficial agentsin a wide range of formulation conditions including concentration,elevated temperatures and duration of stable formulation, thus makingpossible the delivery of beneficial agents in long term implantabledevices that would not otherwise be feasible.

Definitions

As used herein, the following terms have the following meanings:

The term “chemical stability” means that an acceptable percentage ofdegradation products produced by chemical pathways such as oxidation,deamidation, or hydrolysis is formed. In particular, a formulation isconsidered chemically stable if no more than about 35% breakdownproducts are formed after 2 months at 37° C.

The term “physical stability” means that an acceptable percentage ofaggregates (e.g., dimers, trimers and larger forms) are formed by thebeneficial agent. For the formulation (viscous vehicle and beneficialagent) this term means that the formulation retains stability,flowability, and the ability to uniformly dispense the beneficial agent.In particular, a formulation is considered physically stable if no morethan about 15% aggregates are formed after two months at 37° C.

The term “stable formulation” means that at least about 65% chemicallyand physically stable beneficial agent remains after two months at 37°C. (or equivalent conditions at an elevated temperature). Particularlypreferred formulations are those which retain at least about 80%chemically and physically stable beneficial agent under theseconditions. Especially preferred stable formulations are those which donot exhibit degradation after sterilizing irradiation (e.g., gamma, betaor electron beam).

The term “beneficial agent” means peptides, proteins, nucleotides,hormones, viruses, antibodies, etc. that comprise polymers of amino acidor nucleic acid residues. These beneficial agents are generallydegradable in water and generally stable as a dry powder at elevatedtemperatures. Synthetically produced, naturally derived or recombinantlyproduced moieties are included in this term. The term also includeslipoproteins and post translationally modified forms, e.g., glycosylatedproteins. Analogs, derivatives, agonists, antagonists andpharmaceutically acceptable salts of any of these are included in thisterm. The term also includes proteins and/or protein substances whichhave D-amino acids, modified, derivatized or non-naturally occurringamino acids in the D- or L- configuration and/or peptomimetic units aspart of their structure. The term protein will be used in the presentinvention. The term also means that the beneficial agent is present inthe solid state, e.g., powder or crystalline.

The term “excipient” means a more or less inert substance in aformulation that is added as a diluent or vehicle or to give form orconsistency. Excipients are distinguished from solvents such as ETOH,which are used to dissolve drugs in formulations. Excipients includenon-ionic surfactants such as polysorbates, which are used to solubilizedrugs in formulations; preservatives such as benzyl alcohols or methylor propyl parabens, which are used to prevent or inhibit microbialgrowth; chelating agents; flavoring agents; and other pharmaceuticallyacceptable formulation aides.

The term “viscous vehicle” means a vehicle with a viscosity in the rangeof about 1,000 to 10,000,000 poise. The term includes Newtonian andnon-Newtonian materials. Preferred are vehicles with a viscosity ofabout 10,000 to 250,000 poise. The formulations of this invention canuniformly expel beneficial agents suspended in the viscous vehicle fromimplantable drug delivery devices. The formulations exhibit a shear rateat the exit of said devices of 1 to 1×10⁻⁷ reciprocal second, preferablyan exit shear rate of 1×10⁻² to 1×10⁻⁵ reciprocal second.

The term “single phase” means a solid, semi-solid, or liquid homogeneoussystem that is both physically and chemically uniform throughout asdetermined by differential scanning calorimetry (DSC). The DSC scanshould show one peak indicative of a single phase.

The term “biocompatible” means a property or characteristic of a viscousvehicle to disintegrate or break down, over a prolonged period of time,in response to the biological environment in the patient, by one or morephysical or chemical degradative processes, for example by enzymaticaction, oxidation or reduction, hydrolysis (proteolysis), displacement,e.g. ion exchange, or dissolution by solubilization, emulsion or micelleformation, and which material is then absorbed by the body andsurrounding tissue, or otherwise dissipated thereby.

The term “polymer” includes polyesters such as PLA (polylactic acid)[having an inherent viscosity in the range of about 0.5 to 2.0 i.v. ]and PLGA (polylacticpolyglycolic acid) [having an inherent viscosity inthe range of about 0.5 to 2.0 i.v. ], pyrrolidones such aspolyvinylpyrrolidone (having a molecular weight range of about 2,000 to1,000,000), esters or ethers of unsaturated alcohols such as vinylacetate, and polyoxyethylenepolyoxypropylene block copolymers(exhibiting a high viscosity at 37° C.) such as Pluronic 105. Currentlypreferred polymer is polyvinylpyrrolidone.

The term “solvent” includes carboxylic acid esters such as lauryllactate, polyhydric alcohols such as glycerin, polymers of polyhydricalcohols such as polyethylene glycol (having a molecular weight of about200 to 600), fatty acids such as oleic acid and octanoic acid, oils suchas castor oil, propylene carbonate, lauryl alcohol, or esters ofpolyhydric alcohols such as triacetin acetate. Currently preferred islauryl lactate.

The term “surfactant” includes esters of polyhydric alcohols such asglycerol monolaurate, ethoxylated castor oil, polysorbates, esters orethers of saturated alcohols such as myristyl lactate (Ceraphyl 50), andpolyoxyethylenepolyoxypropylene block copolymers such as Pluronic.Currently preferred are gylcerol monolaurate and polysorbates.

The term “antioxidant” means a pharmaceutically acceptable aid forstabilization of the beneficial agent against degradation such asoxidation. Antioxidants include, but are not limited to, tocopherol(vitamin E), ascorbic acid, ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, and propyl gallate. Apreferred antioxidant depends on solubility and the efficiency of theantioxidant for protecting against degradation or chemical change of thebeneficial agent in the preferred vehicle. Currently preferred isascorbyl palmitate.

Preparation of Formulations

The present invention is drawn to stable non-aqueous single phasebiocompatible viscous vehicles capable of suspending beneficial agentsand uniformly dispensing said beneficial agents at body temperatures atlow flow rates over an extended period of time. The present invention isalso directed to formulations containing beneficial agents uniformlysuspended in said single phase biocompatible viscous vehicles which arestable for prolonged periods of time at body temperatures.

Examples of beneficial agents that may be formulated using the presentinvention include those peptides or proteins that have biologicalactivity or that may be used to treat a disease or other pathologicalcondition. They include, but are not limited to, adrenocorticotropichormone, angiotensin I and II, atrial natriuretic peptide, bombesin,bradykinin, calcitonin, cerebellin, dynorphin N, alpha and betaendorphin, endothelin, enkephalin, epidermal growth factor, fertirelin,follicular gonadotropin releasing peptide, galanin, glucagon, GLP-1,gonadorelin, gonadotropin, goserelin, growth hormone releasing peptide,histrelin, human growth hormone, insulin, interferons, leuprolide, LHRH,motilin, nafarerlin, neurotensin, oxytocin, relaxin, somatostatin,substance P, tumor necrosis factor, triptorelin, vasopressin, growthhormone, nerve growth factor, blood clotting factors, ribozymes, andantisense oligonucleotides. Analogs, derivatives, antagonists agonistsand pharmaceutically acceptable salts of the above may also be used.

The beneficial agents useful in the formulations and methods of thepresent invention can be used in the form of a salt, preferably apharmaceutically acceptable salt. Useful salts are known to those ofskill in the art and include salts with inorganic acids, organic acids,inorganic bases, or organic bases.

Beneficial agents that are not readily soluble in non-aqueous solventsare preferred for use in the present invention. One of skill in the artcan easily determine which compounds will be useful on the basis oftheir solubility. The amount of beneficial agent may vary depending onthe potency of the compound, the condition to be treated, the solubilityof the compound, the expected dose and the duration of administration.(See, for example, Gilman, et. al, The Pharmacological Basis ofTherapeutics, 7^(th) ed. (1990) and Remington, Pharmacological Sciences,18^(th) ed. (1990), the disclosures of which are incorporated herein byreference.)

It has been unexpectedly found that using a stable non-aqueous singlephase biocompatible viscous vehicle increases the stability of thebeneficial agent. For example, as seen in FIGS. 1 and 2, human growthhormone (hGH) was found to be stable at 37° C. over 12 weeks informulations of polyvinylpyrrolidone/PEG; Pluronic; and glycerolmonolaurate/lauryl lactate/ polyvinylpyrrolidone. FIG. 1 shows stabilityresults using reverse phase HPLC. FIG. 2 shows stability results usingsize exclusion chromatography.

Generally, stable non-aqueous single phase biocompatiible viscousvehicles may be prepared by combining the dry (low moisture content)ingredients in a dry box or under other dry conditions and blending themat elevated temperature, preferably about 40° C. to about 70° C., toallow them to liquefy. The liquid vehicle is allowed to cool to roomtemperature. Differential scanning calorimetry was used to verify thatthe vehicle was single phase. The final moisture content of the viscousvehicle was <2%.

Generally, the stable formulations of the present invention may beprepared by combining the vehicle and beneficial agent under dryconditions and blending them under vacuum at elevated temperature,preferably about 40° C. to about 70° C., to disperse the beneficialagent uniformly throughout the vehicle. The formulation is allowed tocool to room temperature.

It has been found that drying the beneficial agent prior to formulationenhances the stability of the formulation.

It has also been found that adding antioxidants, such as tocopherol,ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, and propyl gallate reduces the formation of degradationproducts (e.g., unstable chemical intermediates) during sterilization.

Methodology

We have found that stable non-aqueous beneficial agent formulationsutilizing viscous vehicles may be prepared by combining the ingredientsfor the viscous vehicle under dry conditions and blending them atelevated temperature to allow them to liquefy and form a single phase.Once a single phase viscous vehicle is formed, the vehicle is allowed tocool to room temperature. Beneficial agent is added with mixing atelevated temperature under vacuum to uniformly disperse it in theviscous vehicle.

We have tested these beneficial agent formulations, for exampleformulations of hGH, for stability by subjecting them to acceleratedaging tests. Results show that these formulations remained stable overextended periods of time.

We have tested beneficial agent formulations, for example human growthhormone and lysozyme, for stability by suspending them in a variety ofnon-aqueous single phase viscous vehicles prepared according to thepresent invention, then subjecting them to accelerated aging at elevatedtemperatures. The stability of the formulations was measured. Results ofthese studies demonstrate that these formulations were stable atconditions that approximate or exceed storage for one year at 37° C.

We have also tested beneficial agent formulations prepared as describedherein for stability after 2.5 megarads gamma irradiation. Results showthat these formulations remained chemically and physically stable aftersuch irradiation.

Methods

The following methods were used to perform the studies in the Examplesthat follow.

1. Preparing Protein powders

Human Growth Hormone (obtained for example, from BresaGen Limited,Adelaide, Australia)

The active agent was reconstituted in deionized water. The solutioncontaining the active agent was buffer exchanged using an Amicon Diaflo®Ultrafiltration membrane (molecular weight cut-off 10,000).

The diafiltrated active agent solution was spray dried using a Yamatomini-spray dryer. Powder was collected in a collection vessel through acyclone trap. All handling of the spray dried powder took place in a drybox evacuated with nitrogen. The generated powder was analyzed forparticle size and distribution, moisture content, protein content andstability by size exclusion and reverse-phase chromatography.

It is known that the conformation of some proteins can be stabilized bythe addition of a sugar (such as sucrose or mannitol) or a polyol (suchas ethylene glycol, glycerol, glucose, and dextran.)

2. Preparation of Viscous Vehicles

We have found that stable single phase biocompatible viscous vehicle smay be prepared by combining the ingredients and blending them atelevated temperatures to allow them to liquefy and form a single phase.A differential scanning calorimetry scan showed one peak, indicative ofa single phase. The mixing was completed under vacuum to remove trappedair bubbles produced from the powders. The mixer was a dual helix blademixer (D.I.T.) which runs at a speed around 40 rpm. Higher speeds can beused but are not required.

If a three component viscous vehicle is prepared, the solvent portion ofthe vehicle was added to the heated bowl of the mixer first, followed bythe surfactant. The polymer was added last, and the ingredients weremixed until a solution (single phase) resulted. Vacuum was appliedduring mixing to remove air bubbles. The solution was dispensed from thebowl while at elevated temperature, and allowed to cool to roomtemperature. On cooling the vehicle exhibited increased viscosity. Two-and single component gels were made using the same process.

3. Preparation of Beneficial Agent Formulations

To prepare the formulation, the single phase viscous vehicle was heatedand then blended under vacuum with a weigh ed amount of beneficialagent. The beneficial agent and the single phase viscous vehicle wereblended in the same manner as the vehicle was prepared, using a dualhelix blade mixer (or other similar mixer). Mixing speed was between 40and 120 rpm for approximately 15 minutes or until a uniform dispersionwas attained. The resulting mixture was removed from the mixer, sealedin a dry container, and allowed to cool to room temperature.

4. Preparation of Reservoirs

The reservoirs of implantable drug delivery devices (as disclosed inU.S. patent application Ser. No. 08/595,761, incorporated herein byreference) were filled with the appropriate hGH formulation. Theformulation was filled into titanium reservoirs with a polymer plugblocking each end. The filled reservoir was then sealed in a polyfoilbag and placed in a stability testing oven.

It should be noted that the formulations in the reservoirs of thesedevices are completely isolated from the outside environment.

5. Reverse Phase-HPLC (RP-HPLC)

All stability samples of hGH were assayed for protein content andchemical stability by reverse phase chromatography (RP-HPLC). Analyseswere performed on a Hewlett Packard HP-1090 system with a refrigeratedautosampler (4° C.). The chromatographic conditions used are listedbelow.

TABLE 1 RP-HPLC CHROMATOGRAPHIC CONDITIONS Description Parameter ColumnJ. T. Baker-C18, 4.6 × 250 mm Flow Rate 1.0 mL/min. Detection 214 nmMobile Phase A: 0.1% TFA in water B: 0.1% TFA in acetonitrile Gradienttime % A % B  0 65 35  5 50 50 45 35 65 50 30 70 55 65 35

An hGH reference standard solution was prepared and its protein contentcalculated from the absorbance measurement at 280 nm. Three dilutions ofthis solution, representing 80%, 100%, and 120% of the expectedconcentration of hGH in the samples were run in duplicate at thebeginning and the end of each run and used to calculate total proteincontent of the samples.

6. Size Exclusion Chromatography (SEC)

All stability samples of hGH were assayed for protein content and highmolecular weight degradation products by size exclusion chromatography.Analyses were performed on a Hewlett Packard HP-1090 system with arefrigerated autosampler (4° C.). The chromatographic conditions usedare listed below.

TABLE 2 SEC CHROMATOGRAPHIC CONDITIONS Description Parameter ColumnTSK-2000SWXL Flow Rate 0.5 ml/min Detection 214 nm Mobile Phase 25 mMsodium phosphate, 100 mM sodium chloride, pH 7.0

A hGH reference standard solution was prepared and its protein contentcalculated from the absorbance measurement at 280 nm. Three dilutions ofthis solution, representing 80%, 100%, and 120% of the expectedconcentration of hGH in the samples were run in duplicate at thebeginning and the end of each run and used to calculate total proteincontent of the samples. The amount of high molecular weight degradationproducts was calculated by area normalization.

The following examples are offered to illustrate this invention and arenot meant to be construed in any way as limiting the scope of thisinvention.

EXAMPLE 1 Preparation of Non-aqueous Single Phase Viscous Vehicles

The non-aqueous single phase viscous vehicles can be prepared as followsand shown in the below table

A. Glycerol monolaurate (Danisco Ingredients, New Century, Kans.) (25 g)was dissolved in lauryl lactate (ISP Van Dyk Inc., Belleville, N.J.) (35g) at 65° C. Polyvinylpyrrolidone C30 (BASF, Mount Olive, N.J.) (40 g)was added and the mixture blended at about 40 rpm in a dual helix blademixer (D.I.T.) until a single phase was achieved. Trapped air bubbleswere removed by applying vacuum to the mixing chamber. The single phasevehicle was dispensed from the mixer, and allowed to cool to roomtemperature.

B. Glycerol monolaurate (Danisco Ingredients, New Century, Kans.) (25 g)was dissolved in lauryl lactate (ISP Van Dyk Inc., Belleville, N.J.) (35g) at 65° C. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (40 g)was added and the mixture blended at about 40 rpm in a dual helix blademixer (D.I.T.) until a single phase was achieved. Trapped air bubbleswere removed by applying vacuum to the mixing chamber. The single phasevehicle was dispensed from the mixer, and allowed to cool to roomtemperature.

C. Polyvinylpyrrolidone C30 (BASF, Mount Olive, N.J.) (50 g) wasdissolved in polyethylene glycol 400 (Union Carbide) (50 g) atapproximately 65° C. until a single phase solution was formed. Thesingle phase vehicle was dispensed from the mixer, and allowed to coolto room temperature.

D. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (50 g) wasdissolved in polyethylene glycol 400 (Union Carbide) (50 g) atapproximately 65° C. until a single phase solution was formed. Thesingle phase vehicle was dispensed from the mixer, and allowed to coolto room temperature.

E. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (50 g) wasdissolved in castor oil (Spectrum, Gardena, Calif.) (50 g) atapproximately 65° C. until a single phase solution was formed. Thesingle phase vehicle was dispensed from the mixer, and allowed to coolto room temperature.

F. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (50 g) wasdissolved in octanoic acid (Spectrum, Gardena, Calif.) at approximately65° C. until a single phase solution was formed. The single phasevehicle was dispensed from the mixer, and allowed to cool to roomtemperature.

G. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (50 g) wasdissolved in oleic acid (Spectrum, Gardena, Calif.) at approximately 65°C. until a single phase solution was formed. The single phase vehiclewas dispensed from the mixer, and allowed to cool to room temperature.

H. Polyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (35%) wasdissolved in glycerin (Baker, N.J.) (65%) at approximately 65° C. untila single phase solution was formed. The single phase vehicle wasdispensed from the mixer, and allowed to cool to room temperature.

I. Cremophor EL (ethoxylated castor oil) (BASF, Mount Olive, N.J.) (5%)was dissolved in castor oil (Spectrum, Gardena, Calif.)) (70%), andpolyvinylpyrrolidone C17 (BASF, Mount Olive, N.J.) (25%) was added anddissolved by mixing at approximately 40 rpm to form a single phasevehicle. The single phase vehicle was dispensed from the mixer, andallowed to cool to room temperature.

J. Pluronic 105 (BASF, Mount Olive, N.J.) was heated to approximately65° C. with mixing until melted. The single phase vehicle was dispensedfrom the mixer, and allowed to cool to room temperature.

TABLE 3 COMPONENT RATIOS Component Viscosity at Low Polymer SurfactantSolvent Ratio Shear Rate (Poise) PVP GML LL 53:5:42  25,000 PVP GML LL55:10:35 50,000 PVP GML LL 50:15:35  7,000 PVP — LA 60:40 PVP Ceraphyl50 LA 60:10:30 PVP — oleic acid 50:50 30,000 PVP — octanoic acid 55:45 7.000 PVP polysorbate 80 — 50:50 PVP — PEG 400 50:50 PVP castOr oil —50:50 — Pluronic 105 — 100 1,000,000   PVP — glycerin 50:50  5,000Wherein:

-   -   GML=glycerol monolaurate    -   LL=lauryl lactate    -   PVP=polyvinylpyrrolidine C30    -   LA=lauryl alcohol    -   PEG=polyethyleneglycol 400

EXAMPLE 2 Preparation of hGH

A. Preparation by Spray Drying

Lyophilized hGH (BresaGen Limited, Adelaide, Australia) wasreconstituted in 150 ml of deionized water. This stock solutioncontained 1050 mg of hGH. Buffer exchange was accomplished using anAmicon Diaflo® Ultrafiltration membrane (molecular weight cut-off10,000). The ultrafiltration cell was connected to an auxilliaryreservoir containing 5 mM phosphate buffer (pH 7). The cell's fluidvolume, as well as the hGH concentration, remained constant asexcipients were replaced by phosphate buffer.

The diafiltrated protein solution (protein concentration in the solutionapproximately 2%) was spray dried using a Yamato mini-spray dryer.Settings on the spray dryer were as follows: aspiration pressureconstantly adjusted to 1.3 kgf/cm², inlet temperature 120° C., solutionflow rate 2.5 (approximately 3 ml/min). Powder was collected in acollection vessel through a cyclone trap. All handling of the spraydried powder took place in a dry box evacuated with nitrogen (% RH:1-4%). The water content of the suspending vehicles is shown in thetable below.

TABLE 4 WATER CONTENT OF SUSPENDING VEHICLES Water Content of WaterContent of Vehicle Vehicle at T 0 in 12 wks. At 37° C. Vehicle % w/w %w/w Pluronic 105 0.25 0.4 GML/LL/PVP 1.5 1.3 PVP/PEG 2.0 2.0Wherein:

-   -   GML=glycerol monolaurate    -   LL=lauryl lactate    -   PVP=polyvinylpyrrolidine C30    -   PEG =polyethyleneglycol 400

EXAMPLE 3 Preparation of hGH Formulation

A portion of the single phase viscous vehicle was weighed (9 g) andheated to 60° C. hGH (BresaGen Limited, Adelaide, Australia) (1 g) wasadded to the vehicle and mixed for 15 minutes. The mixing was completedunder vacuum to remove air bubbles added from the powder.

Approximately 10 mg of the spray-dried hGH powder were weighed out(content of hGH in the powder was recalculated based on the determinedwater and salt content) and mixed with 100 μl of the vehicle at 55-65°C. (three samples per each vehicle). Special care was taken while mixingpowder in the suspending vehicle to achieve maximum particle uniformdispersion in the vehicle. All steps were done in a dry box.

The resulting suspension was dissolved with 10 ml of release rate bufferand analyzed by size exclusion and reverse-phase chromatography. Spraydried hGH powder was used as a control.

TABLE 5 STABILITY OF hGH SUSPENSIONS AT 37° C. AS MEASURED BY SIZEEXCLUSION CHROMATOGRAPHY Spray-dried PVP/PEG 400 GML/LL/PVP Pluronic 105Time Powder suspension suspension suspension Weeks −80° C. % LS % LS %LS % LS 0 96 ± 1 88 ± 6 92 ± 2 87 ± 7 1 99 ± 8 81 ± 2 94 ± 3 93 ± 3 2 99± 3 83 ± 1 97 ± 1 94 ± 1 3 97 ± 1 84 ± 2 95 ± 2 95 ± 3 4 95 ± 2 82 ± 894 ± 4 93 ± 5 7 95 ± 4 76 ± 3 93 ± 4 88 ± 2 12 97 ± 4 79 ± 3 97 ± 1 95 ±6Each data point represents the mean±relative standard deviation of threeindividual samples taken from three separate vials.

TABLE 6 STABILITY OF hGH SUSPENSIONS at 37° C. AS MEASURED BY REVERSEPHASE CHROMATOGRAPHY spray-dried PVP/PEG 400 GML/LL/PVP Pluronic 105Time Powder suspension suspension suspension Weeks −80° C. % LS % LS %LS % LS 0 104 ± 1 99 ± 3 99 ± 2 89 ± 7 1 104 ± 8 78 ± 2 98 ± 3 96 ± 6 2104 ± 4 73 ± 3 95 ± 1 96 ± 1 3 104 ± 2 78 ± 4 97 ± 3 97 ± 4 4 100 ± 2 74 ± 10 93 ± 4 96 ± 4 7 108 ± 5 72 ± 4 96 ± 2 94 ± 2 9 102 ± 3 66 ± 392 ± 3 93 ± 2 12 101 ± 2 66 ± 1 89 ± 2 92 ± 5Each data point represents the mean±relative standard deviation of threeindividual samples taken from three separate vials.

EXAMPLE 4 Preparation of Reservoirs Release Rate Profiles

Titanium reservoir systems of implantable drug delivery devices (asdisclosed in U.S. patent application Ser. No. 08/595,761, incorporatedherein by reference) were each assembled with an osmotic engine, piston,and rate controlling membrane. The reservoirs were filled with theappropriate amount of viscous vehicle formulation and capped with a flowlug. The systems were placed in a water bath at 37° C., and allowed torelease formulation for an extended period of time. Released materialwas sampled twice per week. Assays for released material were completedusing reverse phase HPLC. The resulting concentrations of beneficialagent for each system were converted to released amount per day. Thebeneficial agent was found to have a zero order release from theimplantable drug delivery device. As shown in FIGS. 3 through 8.

EXAMPLE 5 Stability of hGH in Non-aqueous Viscous Vehicle Formulations

Formulations of 10% w/w hGH in vehicle were prepared as described aboveand placed in vials. The formulations were subjected to acceleratedaging by storing them at elevated temperatures and times shown in thebelow table in a controlled temperature oven.

TABLE 7 % LS by Vehicle Time(hrs) Temperature % LS by SEC RP-HPLCPluronic 105 0 50° C. 98 ± 3 101 ± 3 Pluronic 105 1 50° C. 98 ± 3 101 ±4 Pluronic 105 2 50° C. 100 ± 1  102 ± 3 Pluronic 105 4 50° C. 101 ± 3 105 ± 3 GML/LL/PVP 0 65° C. 99 ± 3 101 ± 3 GML/LL/PVP 1 65° C. 93 ± 6 97 ± 6 GML/LL/PVP 2 65° C. 91 ± 5  95 ± 5 GML/LL/PVP 4 65° C. 95 ± 3 98 ± 3Each data point represents the mean±relative standard deviation of threeindividual samples taken from three separate vials.

Results, presented in the following table, demonstrate that theseformulations were able to maintain the stability of the hGH in eachcase. In each case, at least 70% hGH was retained.

TABLE 8 RECOVERY OF hGH FROM NONAQUEOUS SUSPENSIONS Vehicle % LS byRP-HPLC % LS by Size-exclusion HPLC PVP/PEG 400 99 ± 3% 88 ± 6%GML/LL/PVP 99 ± 2% 92 ± 2% Pluronic 105 89 ± 7% 87 ± 7%Each data point represents the mean±relative standard deviation of threeindividual samples taken from three separate vials. %LS or % labelstrength (measured protein content+theoretical protein content)×100%

Modification of the above-described modes of carrying out variousembodiments of this invention will be apparent to those of skill in theart following the teachings of this invention as set forth herein. Theexamples described above are not limiting, but are merely exemplary ofthis invention, the scope of which is defined by the following claims.

1. A stable non-aqueous single-phase biocompatible viscous vehicle,comprising: a polymer consisting of polyvinylpyrrolidone; a surfactantconsisting of glycerol monolaurate; and a solvent consisting of laurellacate; wherein the viscosity of the vehicle is between about 1,000 andabout 10,000,000 poises.
 2. A stable non-aqueous single-phasebiocompatible viscous vehicle comprising: a polymer consisting ofpolyvinylpyrrolidone; a surfactant consisting of polysorbate; and asolvent consisting of lauryl lactate; wherein the viscosity of thevehicle is between about 1,000 and about 10,000,000 poise.
 3. Anon-aqueous formulation comprising at least one beneficial agentuniformly suspended in a non-aqueous single-phase biocompatible viscousvehicle, the non-aqueous single-phase biocompatible viscous vehiclecomprising: a polymer consisting of glycerol monolaurate or polysorbate;and a solvent consisting of lauryl lactate.
 4. A stable non-aqueousviscous protein formulation that is capable of being uniformly dispensedover an extended period of time at a low flow rate and is stable at bodytemperature for extended periods of time, the stable non-aqueous viscousprotein formulation comprising: a) at least one protein; and b) anon-aqueous single-phase biocompatible viscous vehicle comprising apolymer consisting of polyvinylpyrrolidone, a surfactant consisting ofglycerol monolaurate or polysorbate, and a solvent consisting of lauryllactate.
 5. The stable non-aqueous viscous protein formulation of claim4, wherein the at least one protein is present in an amount of at leastabout 0.1% (w/w).
 6. The stable non-aqueous viscous protein formulationof claim 4, wherein the at least one protein is present in an amount ofat least about 10% (w/w).
 7. The formulation of claim 4, wherein thestable non-aqueous viscous protein formulation is capable of beinguniformly dispensed over an extended period of time at a low flow rateand is stable at 65° C. for at least about 2 months.
 8. The formulationof claim 4, wherein the stable non-aqueous viscous protein formulationis capable of being uniformly dispensed over an extended period of timeat a low flow rate and is stable at 37° C. for at least about 3 months.9. The formulation of claim 4, wherein the viscosity of the vehicle isbetween about 1,000 and about 10,000,000 poise.
 10. The formulation ofclaim 4, wherein the beneficial agent is dried to a low moisture contentprior to incorporated in the stable non-aqueous viscous proteinformulation.
 11. A method for preparing a stable non-aqueoussingle-phase biocompatible viscous vehicle, the method comprising: (1)selecting a polymer consisting of polyvinylpyrrolidone, a surfactantconsisting of glycerol monolaurate or polydsorbate, and a solventconsisting of lauryl lactate; (2) blending the polymer, the surfactant,and the solvent at elevated temperature under dry conditions to allowthe polymer, the surfactant, and the solvent to liquefy; and (3)allowing the liquefied components to cool to room temperature such thata stable non-aqueous single-phase biocompatible viscous vehicle isformed.
 12. The method of claim 11, wherein the viscosity of the vehicleis between about 1,000 and about 10,000,000 poise.
 13. The method ofclaim 11, wherein the at least one beneficial agent is present in anamount of at least about 0.1%° (w/w).
 14. The method of claim 11,wherein the at least one beneficial agent is present in an amount of atleast about 10%°(w/w).